Variants of glycogen synthase kinase 3 and uses thereof

Abstract

The present invention provides GSK3β variants, including splice variants expressed in bone or other tissues. These variants have a broad range of functional activity, from inhibiting the Wnt pathway to activating the pathway as dominant-negatives of the full-length GSK3β. These variants, as well as their modulators, can be used to treat diseases that involve GSK3β or its associated signaling pathways.

1 . An isolated protein comprising: 
 (a) a human glycogen synthase kinase 3β (GSK3β) amino acid sequence in which five or more consecutive amino acids encoded by a GSK3-WT exon are absent, wherein the GSK3-WT exon is selected from the group consisting of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 11; or    (b) an amino acid sequence at least 90% identical to (a).    
     
     
         2 . The isolated protein of  claim 1 , wherein the isolated protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 16, 17, 19 and 20.  
     
     
         3 . The isolated protein of  claim 1 , wherein the GSK3-WT exon is selected from the group consisting of exon 7, exon 8, and exon 9 and wherein five or more consecutive amino acids encoded by GSK3-WT exon 10 are also absent.  
     
     
         4 . The isolated protein of  claim 3 , wherein the isolated protein is selected from the group consisting of SEQ ID NOs: 12, 14, 19 and 20.  
     
     
         5 . A method of inhibiting an activity of glycogen synthase kinase 3β (GSK3β) in human cells, said method comprising introducing the isolated protein of  claim 3  into said cells such that the activity of GSK3β in said cells is inhibited.  
     
     
         6 . The method of  claim 5 , wherein said human cells are bone cells, neuronal cells, adipose tissue cells, or metabolic tissue cells.  
     
     
         7 . A pharmaceutical composition comprising the isolated protein of  claim 3 .  
     
     
         8 . An antibody capable of binding to the amino acid sequence in the isolated protein of  claim 2 , but not to the amino acid sequence depicted in SEQ ID NO:10.  
     
     
         9 . An isolated polynucleotide comprising an open reading frame encoding the isolated protein of  claim 1 .  
     
     
         10 . An isolated polynucleotide capable of inhibiting the expression of a GSK3β splice variant by RNA interference or an antisense mechanism, wherein said isolated polynucleotide comprises a sequence which is complementary or antisense to at least a portion of an mRNA encoding said amino acid sequence in the isolated protein of  claim 2 , but not to mRNAs encoding GSK3-WT.  
     
     
         11 . A method of identifying agents capable of modulating a biological activity of the isolated protein of  claim 2 , said method comprising detecting said biological activity in the presence or absence of a candidate molecule, wherein a change in said biological activity in the presence of said candidate molecule as compared to that in the absence of said candidate molecule is indicative that said candidate molecule is capable of modulating said biological activity.  
     
     
         12 . An agent identified by the method of  claim 11 , wherein said agent is capable of inhibiting a dominant-negative effect of the isolated protein on the activity of a full-length GSK3β in Wnt signaling, and wherein the isolated protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 19 and 20.  
     
     
         13 . A method for inhibiting GSK3β activity in human cells, said method comprising introducing into said cells an agent selected from the group consisting of: 
 an antibody capable of binding to a protein encoded by a GSK3β splice variant, said GSK3β splice variant being selected from the group consisting of GSK3Δ8 and GSK3Δ7-9, and said antibody being incapable of binding to a full-length GSK3-WT protein;    an antisense molecule for said GSK3β splice variant, but not for GSK3-WT;    an expression vector encoding said antisense molecule;    an RNAi molecule for said GSK3β splice variant, but not for GSK3-WT;    one or more expression vectors encoding said RNAi molecule; and    an expression vector comprising an open reading frame encoding the protein of  claim 3 .    
     
     
         14 . The method of  claim 13 , wherein said human cells are bone cells, neuronal cells, adipose tissue cells, or metabolic tissue cells.  
     
     
         15 . A method for inhibiting GSK3β activity in human bone cells, said method comprising introducing into said cells an agent selected from the group consisting of: 
 an antibody capable of binding to a protein encoded by a GSK3β splice variant, said GSK3β splice variant being selected from the group consisting of GSK3-8b and GSK3Δ10, and said antibody being incapable of binding to a GSK3-WT protein;    an antisense molecule for said GSK3β splice variant, but not for GSK3-WT;    an expression vector encoding said antisense molecule;    an RNAi molecule for said GSK3β splice variant, but not for GSK3-WT; and    one or more expression vectors encoding said RNAi molecule.    
     
     
         16 . A method for treating a disease that is responsive to modulation of a GSK3β-associated signaling pathway, said method comprising administering a pharmaceutical composition to a patient who has said disease, said pharmaceutical composition comprising an agent selected from the group consisting of: 
 an antibody capable of binding to a protein encoded by a GSK3β splice variant, said GSK3β splice variant being selected from the group consisting of GSK3Δ8 and GSK3Δ7-9, and said antibody being incapable of binding to a GSK3-WT protein;    an antisense molecule for said GSK3β splice variant, but not for GSK3-WT;    an expression vector encoding said antisense molecule;    an RNAi molecule for said GSK3β splice variant, but not for GSK3-WT;    one or more expression vectors encoding said RNAi molecule;    a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 19 and 20; and    an expression vector comprising an open reading frame encoding said polypeptide.    
     
     
         17 . The method of  claim 16 , wherein said disease is a bone loss disease, a neurodegenerative disease, or a metabolic disease.  
     
     
         18 . The method of  claim 16 , wherein said disease is osteoporosis, Type 2 diabetes, obesity or Alzheimer's disease.  
     
     
         19 . A method of treating a disease characterized by bone loss, said method comprising introducing a pharmaceutical composition into bone cells of a patient who has said disease, wherein said pharmaceutical composition comprises an agent selected from the group consisting of: 
 an antibody capable of binding to a protein encoded by a GSK3β splice variant, said splice variant being selected from the group consisting of GSK3-8b and GSK3Δ10, and said antibody being incapable of binding to a GSK3-WT protein;    an antisense molecule for said splice variant, but not for GSK3-WT;    an expression vector encoding said antisense molecule;    an RNAi molecule for said splice variant, but not for GSK3-WT; and    one or more expression vectors encoding said RNAi molecule.    
     
     
         20 . A method of identifying agents capable of inhibiting a biological activity of GSK3-8b, said method comprising detecting said biological activity in the presence or absence of a candidate molecule, wherein a reduction in said biological activity in the presence of said candidate molecule as compared to in the absence of said candidate molecule is indicative that said candidate molecule is an inhibitor of GSK3-8b.  
     
     
         21 . An isolated protein comprising an amino acid sequence which includes: 
 a sequence segment of amino acids 235-240 of GSK3-6b;    a sequence segment of amino acids 269-278 of GSK3-7b;    a sequence segment of amino acids 269-274 of GSK3Δ8;    a sequence segment of amino acids 235-242 of GSK3Δ7-9;    a sequence segment of amino acids 204-240 of GSK3Δ6-10; or    a sequence segment of amino acids 76-82 of GSK3Δ2-11,    wherein each said sequence segment comprises at least five amino acid residues.    
     
     
         22 . An isolated polynucleotide comprising an open reading frame encoding said isolated protein of  claim 21.